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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
  • C12N15/73—Expression systems using phage (lambda) regulatory sequences
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C12P21/00—Preparation of peptides or proteins
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/10—Plasmid DNA
  • C12N2800/106—Plasmid DNA for vertebrates
  • C12N2800/107—Plasmid DNA for vertebrates for mammalian
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/24—Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication

Definitions

• the present invention relates to a method of maintaining and producing plasmids in a gram negative bacterium without the use of antibiotic selection pressure. Further, the invention relates to the drugless plasmids produced as well as formulations and/or compositions containing the drugless plasmids, and formulations and/or compositions containing the protein or immunogen produced using the drugless plasmids, and to methods of administering the formulations and/or compositions to a host. The invention relates to gram negative bacteria containing the drugless plasmids. BACKGROUND OF THE INVENTION
• Plasmids are extra-chromosomal DNA molecules that are separate from the chromosomal DNA and are capable of replicating independently of the chromosomal DNA (Lipps G. (editor). (2008). Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6). Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms. They are considered transferable genetic elements, or replicons, capable of autonomous replication within a suitable host. Plasmids are naked DNA and do not encode genes necessary to encase the genetic material for transfer to a new host. Therefore host-to-host transfer of plasmids requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation (Lipps G. 2008).
• Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. For example, plasmids may carry genes that provide resistance to naturally occurring antibiotics.
• the proteins produced by the plasmid may act as toxins which also provide a selective advantage under a given environmental state. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage under conditions of nutrient deprivation (Lipps G., 2008).
• plasmids are prepared wherein a gene is inserted that generates a protein which makes cells resistant to a particular antibiotic.
• the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant.
• Genes of interest may be delivered using a plasmid containing an antibiotic -resistant marker.
• a gene is typically inserted at a multiple cloning site (MCS, or polylinker).
• MCS multiple cloning site
• the antibiotic -resistant genes are expressed and the expressed proteins break down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often /using the alkaline lysis method) to isolate the plasmid of interest.
• antibiotics in medicine has now become a problem. Many bacteria, including pathogens of infectious diseases, are already resistant and can no longer be controlled with the particular antibiotic. Antibiotics have been used too frequently in human and animal medicine. Moreover, of even far greater significance is the fact that for a long time they were added to animal feed as a performance enhancer. This practice is now largely outlawed, but the pervasive antibiotics have given a survival advantage to those bacteria that have a corresponding resistance gene. Moreover, resistance genes in bacteria are often located on mobile DNA units, which can be exchanged between different species.
• genes may have a considerable impact on the plasmid production process, as constitutive expression of these genes imposes an unnecessary metabolic load on the bacterial host cell (Cranenburgh, R. M., et al., Nucleic Acids Res. 2001, 29, e26; Rozkov, A., et al., Enzyme Microb. Technol. 2006, 39, 47-50). Reducing the size of the plasmids by eliminating these genes would lead to improved stability and yield of pDNA obtained by the fermentation process (Smith, M. A., et al., Can. J. Microbiol. 1998, 44, 351-355).
• Cranenburgh, R.M. et al. reported the construction of two novel Escherichia coli strains (DHllacdapD and DHllacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter (Cranenburgh, R. M., et al., 2001).
• Mairhofer et al. recently investigated designing bacterial host strains that serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid.
• Several bacterial strains were modified, so that the plasmid' s replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction (Mairhofer, J. et al, Biotechnol. J., 3, 83-89, 2008).
• An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression (Mairhofer, J. et al., 2008).
• RNA I Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed.
• the authors reported that different commercially available plasmids could be selected by various modified Escherichia coli strains. They further designed a minimalistic plasmid devoid of any selection marker.
• Antibiotic-free plasmids as well as gram-negative bacteria comprising the antibiotic- free plasmids are provided.
• Compositions comprising the antibiotic-free plasmids comprising a heterologous gene encoding an immunogen or a protein and compositions comprising the immunogen or the protein expressed using the antibiotic-free plasmids are also provided.
• the gram-negative bacteria are engineered to contain one or more heterologous polynucleotide in the nonessential region of the bacterial chromosome.
• the antibiotic-free plasmids comprise a polynucleotide encoding a repressor which regulates the expression of the heterologous polynucleotide on the bacterial chromosome.
• the antibiotic-free plasmids may further comprise one or more polynucleotide encoding an immunogen or a protein.
• Methods of the invention include methods of producing antibiotic-free plasmids and methods for transfer of foreign genes into mammalian cells using the antibiotic-free plasmids.
• Figure 1 describes the first component of the drugless concept which is the cloning of the sacB gene coding for levansucrase in gram-negative bacteria that leads to the rapid death of the transformed bacteria when plated on a medium containing sucrose.
• Figure 2 describes the second and third components of the drugless or antibiotic-free concept which is the immunity region of phage ⁇ , wherein cl codes for cl repressor; cro codes for Cro protein; N codes for the transcription anti-terminator; ell codes for cl activator; cIII codes for the cIII protease inhibitor; OL 1, 2 and 3 and OR 1,2 and 3 are operators; PL and PR are the rightward and leftward promoters; PRE is the promoter for repressor establishment; PRM is the promoter for repressor maintenance.
• Figure 3 describes the theoretical overview of the "drugless” concept wherein the cl repressor activity, expressed from the pDLl (DrugLess Plasmid) plasmid, inhibits transcription of the toxic sacB gene product placed under the ⁇ Pr promoter located on the cell host chromosome and wherein the host E. coli cells' viability in the presence of sucrose is ensured by a sufficient expression level of the ⁇ cl repressor protein from the plasmid.
• the cl repressor activity expressed from the pDLl (DrugLess Plasmid) plasmid, inhibits transcription of the toxic sacB gene product placed under the ⁇ Pr promoter located on the cell host chromosome and wherein the host E. coli cells' viability in the presence of sucrose is ensured by a sufficient expression level of the ⁇ cl repressor protein from the plasmid.
• Figure 4 describes the "sucrose sensitivity" of the inactivated system wherein a switch to 42°C in temperature inhibits the binding activity of cl repressor to the ⁇ Pr promoter and renders the host cells sensitive to sucrose.
• FIG. 5 illustrates the combination of the host strain with the antibiotic-free pDLl plasmid, resulting from the transformation with pDLl, and growth in medium containing sucrose leading to drugless plasmid yield.
• Figures 6A-6C illustrate the experimental plan.
• FIG 6A provides an overview
• FIG 6B illustrates that the ApurN (or AedA) parental strain is resistant to kanmaycin and expresses constitutively SacB + conferring its sensitivity to sucrose when the cells are incubated at 30°C-37°C.
• the ApurN (or AedA) parental strain are transformed with either the pPB829 or pPB838 plasmids, these cells gain the ability to survive and grow in the presence of sucrose when incubated at 30-37 0 C
• FIG 6C illustrates that the ApurN (or AedA) parental strain transformed with either the pPB829 or pPB838 plasmids (cl plasmids) is able to grow in LB medium containing either kanamycin, sucrose or chloramphenicol as well.
• FIG. 7 describes both plasmids (pPB829 and pPB838) marked with the genes encoding chloramphenicol acetyl transferase (Cat R ) and containing the ⁇ cl gene.
• pB838 is a derivative of pMCS5 in which Chloramphenicol acetyl transferase gene (cat) replaces the ampicillin resistance gene.
• This plasmid contains the cl gene placed under the control of the weak promoter of Kanamycin gene (Pl).
• pPB829 is a pVR1012 plasmid derivative containing the cat gene.
• the plasmids contain the cl gene which is placed under the control of the weak promoter (Pl). When introduced in the ApurN (or AedA) parental E. coli strain, these plasmids will enable the cells to grow in the presence of sucrose. Chloramphenicol allows demonstration of the proof of concept.
• FIGs 8A-E illustrate host strain engineering.
• FIGs 8A and 8B illustrate the generation of the ⁇ Pr::sacB Qkan cassette to be inserted by allelic replacement of edA or purN gene, respectively, into the E. coli chromosome.
• Figure 8C illustrates the engineering of the full sacB cassette ⁇ ApurN ⁇ ⁇ Prr.sacB cat) by PCR and joining PCR used for the allelic replacement of the edA gene into the ApurN ' ⁇ ⁇ Pr::sacB Km E. coli host strain for the generation of the double sacB cassette E. coli strain.
• FIG. 9A illustrates in part the two PCR products (respectively 151 and 729 bp) that were purified and used as a template in a second PCR step with the PBl 186 and PBl 189 primers and the Phusion DNA polymerase.
• FIG. 9B illustrates the PCR check showing that the ⁇ Pr: :sacB + ⁇ .Kan cassette is introduced into the chromosome of E. coli.
• FIG. 9A illustrates in part the two PCR products (respectively 151 and 729 bp) that were purified and used as a template in a second PCR step with the PBl 186 and PBl 189 primers and the Phusion DNA polymerase.
• FIG. 9B illustrates the PCR check showing that the ⁇ Pr: :sacB + ⁇ .Kan cassette is introduced into the chromosome of E. coli.
• FIG. 9C illustrates that the ⁇ Pr: :sacB + ⁇ .Kan cassette is correctly inserted at the locus by allelic replacement of either edA and/or purN gene(s).
• Figure 9D illustrates the use of PCR to demonstrate the dual sacB cassette host strain engineering, namely the deletions of both edA and purN in E. coli by allelic replacement. The PCR check shows that both ⁇ Pr::sacB + ⁇ .Kan and ⁇ Pr::sacB + ⁇ .Cat cassettes are correctly introduced at the specific loci into the chromosome of E. coli, as confirmed by sequencing.
• Figure 1OA demonstrates that the host strain (ApurN ⁇ Pr: :sacB + ⁇ kan) is highly sensitive (no growth) to sucrose, that cells transformed either with pPB829 or pPB838 plasmid grew well in the presence of sucrose at 30°C, that sacB gene expression was perfectly repressed by the cl gene product synthesized from each cl plasmid and that the plasmid maintenance was 100% efficient as clearly demonstrated by the control experiment performed in parallel with similar cells growing in the presence of chloramphenicol. Raising the temperature to 42°C caused inactivation of the cl gene product and death of the cells plated on LB agar containing either sucrose or chloramphenicol.
• FIGs lOB-C demonstrate that the dual sacB cassettes E. coli strain (ApurN ⁇ Pr::sacB + ⁇ kan AedA ⁇ Pr: : sacB + ⁇ Cat) is highly sensitive (no growth) in the presence of sucrose when incubated at 3O 0 C and 42 0 C while some spontaneous mutants appear with the one sacB cassette E. coli strain plated on sucrose.
• FIG 1OC also demonstrates the lowest sucrose concentration required for the dual sacB cassettes E. coli strain compared to the one sacB cassette E. coli strain (from 10% to 2% final).
• Figure 11 illustrates the stability of the pPB829 and pPB838 plasmids in growing cells.
• Figure 12A shows a lack of spontaneous mutation in the ⁇ Prr.sacB one sacB cassette at 30°C.
• Figures 12B-C demonstrate that the presence of two sacB cassettes (rather than one) confers a better robustness to sucrose sensitivity (still optimal at 2% sucrose) at temperatures ranging from 3O 0 C to 37 0 C.
• the presence of one sacB cassette on the E. coli chromosome was less robust at sucrose concentration ranging from 2% to 4% at similar temperatures.
• the mutation rate to sucrose with the two sacB cassettes E. coli strain was undetectable at the lowest sucrose concentration (2%) when incubated at 37 0 C while the mutation rate with the one sacB cassette E.
• coli strain was ranging from 3.8 xlO "6 to 5 xlO "5 .
• An independent set of experiment showed that the mutation rate in the two sacB cassettes E. coli strain was nearly 5 xlO "10 when incubated at 37 0 C in the presence of 2% sucrose.
• Figures 13A-B show the experimental procedures to enable the screening of the transformed cells by the chloramphenicol (Cat) marked cl plasmids without the use of antibiotic as selection pressure on agar plates.
• Figures 14A-C illustrate the efficacy of the experimental procedure to select transformed cells with the cl plasmid only (pPB829 and pPB838) without the use of antibiotic as selection pressure.
• Figures 15A-B illustrate the stability and yield of the pPB829 and pPB838 plasmids in the growing E .coli cells which contain one copy of the sacB cassette.
• Figures 15C-E illustrate the stability and yield of cat-free pPB885 plasmid in both one and two sacB cassettes E. coli strain (ApurN ⁇ Pr::sacB + ⁇ kan and ApurN ⁇ Pr::sacB + ⁇ kan AedA ⁇ Pr: : sacB + ⁇ cat, respectively).
• Figure 15F illustrates the plasmid transfection efficacy (GFP expression) in CHO cells of the antibiotic-free plasmid (pPB896 /Sucrose as selection pressure) and antibiotic plasmid (pCG105 / Cat as selection pressure). No obvious difference is observed in terms of expressed GFP protein between these two selection pressure (Sucrose versus Cat as antibiotic).
• Figure 16 shows the pCRblunt + PBl 184-1185 plasmid map.
• Figure 17 shows the restriction map and characteristics of pPB828.
• Figure 18 shows the restriction map and characteristics of pPB829.
• Figure 19 shows the plasmid map and characteristics of pPB838.
• Figure 20 shows the plasmid map and characteristics of pPB844.
• Figure 21 shows the plasmid map and characteristics of pPB845.
• Figure 22 shows the plasmid map and characteristics of pPB846.
• Figure 23 shows the plasmid map and characteristics of pPB847.
• Figure 24 shows the plasmid map of pPB885.
• the pPB885 feature map shows: two CDS: cl repressor: 324-1037; AP(R): 2321-2990 (complementary), original location
• Figure 25 shows the plasmid map of the pPB896.
• the pPB896 feature map shows: three CDS: GFP: 1675-2397 (original location 1676...2398); cl repressor: 4089-4802;
• Cm(R): 4828-5484 (complementary), complement (950-1606); one intron: intron A: 805- 1625 (original location 805-1625); one eukaryotic promoter: CMV promoter: 1-683; one replication origin: 3133-3640 (original location 2425..2932); one terminator: BGH transcriptional terminator/PolyA: 2405-2951 (original location 1697..2243); one 5' UTR: CMV IE 5' UTR: 684-804 (original location 684..804).
• Figure 26 shows the sequence alignment of cl repressor proteins and sequence identity percentage.
• Figure 27 shows the sequence alignment of sacB proteins and sequence identity percentage.
• Figure 28 is a table showing the SEQ ID NO assigned to the polynucleotide and protein.
• Figure 29 shows the sequence alignment of the polynucleotides encoding cl repressor proteins and sequence identity percentage at the nucleotide level.
• Figure 30 shows the sequence alignment of the polynucleotides encoding sacB proteins and sequence identity percentage at the nucleotide level.
• Figure 31 shows the sequences for Pl promoter, cl native promoter, ⁇ Pr promoter, ⁇ Pr promoter+5' UTR+sacB gene, Pl promoter + cl gene, and cl native promoter + cl gene.
• the animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g., pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and fish.
• the term "animal” also includes an individual animal in all stages of development, including embryonic and fetal stages.
• protein protein
• peptide polypeptide
• polypeptide fragment polymers of amino acid residues of any length.
• the polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
• nucleic acid or “polynucleotide” is used interchangeably and refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
• polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
• the sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component.
• Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support.
• the polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
• drugless plasmid or “antibiotic-free plasmid” is used interchangeably and refers to a DNA plasmid which does not contain an antibiotic selection gene.
• gene is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
• the subject matter disclosed herein relates to a novel approach to generating bacterial plasmid DNA vectors by circumventing the use of antibiotic resistance genes to produce safer vaccines and immunogenic compositions.
• the subject matter disclosed herein demonstrates a new concept for maintaining a high number of plasmid copies within a gram negative bacterium host cell which is based on three components.
• the first component is a gram negative bacterium host which expresses a toxic product for the bacterium under defined culture conditions wherein the toxic gene is inserted into a non-essential region of the bacterium chromosome.
• the second component is the presence of a gene on the gram negative bacterium chromosome wherein the gene encodes a toxic product under the control of a constitutive promoter that can be tightly regulated.
• the third component is the expression of a specific repressor from a plasmid that regulates the promoter operably linked to the toxic gene on the host chromosome.
• the gram negative bacteria contemplated in the present invention include, but are not limited to, Avibacterium, Brucella, Escherichia coli, Haemophilus (e.g., Haemophilus suis), Salmonella (e.g., Salmonella enteridis, Salmonella typhimurium, Salmonella infantis), Shigella, Pasteurella, and Rimeirella.
• the toxic gene is a structural sacB gene that encodes levansucrase.
• the toxic gene is a structural sacB gene of Bacillus subtilis which encodes the Bacillus subtilis levansucrase. Expression of sacB in its natural environment is harmless to gram positive bacterium but when expressed in gram negative bacterium, it leads to rapid death of the transformed bacterium when they are plated on a medium containing sucrose.
• the present invention provides a sacB protein (levansucrase).
• the present invention provides a sacB protein having a sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74, and variant or fragment thereof.
• the present invention provides a sacB protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74.
• the present invention provides fragments and variants of the sacB protein identified above (SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74) which may readily be prepared by one of skill in the art using well-known molecular biology techniques.
• Variants are homologous polypeptides having an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the polypeptides of the invention, particularly to the amino acid sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74.
• the present invention provides a polynucleotide, such as a sacB gene, encoding a sacB protein (levansucrase), for example, a polynucleotide encoding a sacB protein having a sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74.
• a polynucleotide such as a sacB gene, encoding a sacB protein (levansucrase), for example, a polynucleotide encoding a sacB protein having a sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74.
• the present invention provides a polynucleotide encoding a sacB protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NO:4, 60, 62, 64, 66, 68, 70, 72 or 74, or a conservative variant, an allelic variant, a homolog or a fragment comprising at least eight or at east ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.
• the present invention provides a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:3, 61, 63, 65, 67, 69, 71, or 73, or a variant thereof.
• the present invention provides a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to one of a polynucleotide having a sequence as set forth in SEQ ID NO:3, 61, 63, 65, 67, 69, 71, or 73, or a variant thereof.
• toxic genes which may be utilized in the present invention include, but are not limited to, Listeria or Staphylococcus genes.
• a toxic gene can be a DNA that is expressed as a toxic gene product (a toxic protein or RNA), or can be toxic in and of itself. Examples of such toxic gene products are well known in the art, and include, but are not limited to, restriction endonucleases (e.g., Dpnl), CcdA/CcdB (Maki S. et al. J. MoI. Biol. 256: 473-482, 1996) and genes that kill hosts in the absence of a suppressing function, e.g. kicB.
• a toxic gene can alternatively be selectable in vitro, e.g., a restriction site.
• the term "homologs" includes orthologs, analogs and paralogs.
• Analogs, orthologs, and paralogs of a wild-type polypeptide can differ from the wild-type polypeptide by post-translational modifications, by amino acid sequence differences, or by both.
• homologs of the invention will generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98% , 99% sequence identity, with all or part of the wild-type polypeptide or polynucleotide sequences, and will exhibit a similar function.
• a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
• a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
• Variants of a particular polynucleotide of the invention can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
• "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein.
• Variant proteins encompassed by the present invention are biologically active, that is they the ability to elicit an immune response.
• Variants include allelic variants.
• allelic variant refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1- 5% variance in a polynucleotide or a polypeptide.
• Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same genetic locus in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention.
• substitution denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue.
• particularly preferred substitutions will generally be conservative in nature, as described above.
• polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, e.g., optimized codon usage for a specific host.
• optimized refers to a polynucleotide that is genetically engineered to increase its expression in a given species.
• the DNA sequence of the polypeptide can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucleotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites.
• Increased expression of the polypeptide of the present invention can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species.
• the term "frequency of preferred codon usage” refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is functionally unchanged.
• RNA sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman).
• Wilbur and Lipman Wilbur and Lipman algorithm
• RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
• RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
• the sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucleotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA).
• the promoter which is operably linked to the toxic gene of the present invention is a promoter from ⁇ phage
• ⁇ phage is a temperate phage that lives in E. coll. Once the phage is inside its host, it may integrate itself into the host's DNA. In this state, ⁇ is called a prophage and stays resident within the host's genome, without causing much harm to the host. This way, the prophage gets duplicated with every cell division of the host.
• the DNA of the prophage that is expressed in this state codes for proteins that look out for signs of stress in the host cell. Stress can be a result of starvation, poisons (like antibiotics), and other factors that can damage or destroy the host.
• the prophage When the stress condition is detected, the prophage becomes active again, excises itself from the DNA of the host cell and enters its lytic cycle. The reactivated phage takes apart the host's DNA and reprograms its protein factory to produce new phages in multiple copies. When all resources of the host are depleted from building new phages, the cell is lysed (the cell membrane is broken down), and the new phages are released.
• the lambda repressor gene system consists of (from left to right on the chromosome): c/ gene, OR3, OR2, ORl, cro gene, c/ gene codes for the ⁇ repressor ("the cl repressor protein").
• the region of the genome that codes for the cl repressor protein is known as the immunity region.
• the cl repressor protein is both a positive and a negative regulator of gene transcription. It allows the phage ⁇ to stay "latent" on the chromosome of its host bacterium.
• the lysogenic state of the ⁇ bacteriophage is maintained by binding of regulatory protein cl to the OR (right operator) and OL (left operator) operators from ⁇ Pl and ⁇ Pr promoters, respectively, preventing transcription of the proteins necessary for the lytic stage.
• Bacterial cells harboring a lysogenic ⁇ phage are immune to further infection by ⁇ phage.
• the cl repressor protein inhibits the lytic development of any additional infecting phage particles.
• the promoter comprises a polynucleotide having a sequence as set forth in SEQ ID NO:5.
• the promoter comprises a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having a sequence as set forth in SEQ ID NO:5.
• the repressor protein of the present invention is a cl repressor protein, such as the cI857 repressor which is temperature sensitive. A ⁇ phage carrying cI857 as a lysogen will grow at temperatures below 39°C but will then induce lytic growth by an increase in temperature.
• the cl repressor protein is active and binds to the right and left operators of the infecting phage. This prevents transcription of any phage proteins and thus prevents lysis. However, at 42 0 C, the cl repressor is inactivated and cannot bind the promoter operators.
• the present invention provides a cl repressor protein.
• the present invention provides a cl repressor protein having a sequence as set forth in SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58, and variant or fragment thereof.
• the present invention provides a cl repressor protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to the polypeptide having a sequence as set forth in SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58.
• the present invention provides fragments and variants of the cl repressor protein identified above (SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58) which may readily be prepared by one of skill in the art using well-known molecular biology techniques.
• Variants are homologous polypeptides having an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the polypeptides of the invention, particularly to the amino acid sequence as set forth in SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58.
• the present invention provides a polynucleotide, such as a cl gene, encoding a cl repressor protein, such as a polynucleotide encoding a cl repressor protein having a sequence as set forth in SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58.
• the present invention provides a polynucleotide encoding a cl repressor protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NO:2, 44, 46, 48, 50, 52, 54, 56, or 58, or a conservative variant, an allelic variant, a homolog or a fragment comprising at least eight or at east ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.
• the present invention provides a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1, 45, 47, 49, 51, 53, 55, 57, or 59, or a variant thereof.
• the present invention provides a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having a sequence as set forth in SEQ ID NO: 1, 45, 47, 49, 51, 53, 55, 57, or 59, or a variant thereof.
• the promoter driving the cl gene is the native cl gene promoter. In another embodiment, the promoter driving the cl gene is the weak promoter of Kanamycin gene (Pl). In another embodiment, the promoter comprises a polynucleotide having a sequence as set forth in SEQ ID NO:29, 83, or 30. In yet another embodiment, the promoter comprises a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having a sequence as set forth in SEQ ID NO:29, 83, or 30.
• promoter/repressor pairs which may be utilized in the methods of the present invention include, but are not limited to, a carB promoter controlled by the CarA-CarS repressor-antirepressor pair, growth hormone gene promoters and repressors and Lac repressor (lad) and the Ptrc-2 promoter.
• mutant promoter/repressor pair to serve as the substrate for the mutagenesis may be selected from among such mutants presently available and described in the art.
• Studies of the binding of mutant repressors to their operators See, e.g., Nelson and Sauer, Cell, 42:549 (1985); Nelson and Sauer, J. MoI. Biol. 192:27 (1986); and Gussin, et al, Lambda II, (Hendrix, Roberts, Stahl, and Weisberg, eds) Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 93-123 (1983)] permit the selection of known mutant promoter and/or mutant repressor sequences which may provide reduced, but not abolished, transcriptional rates.
• This selected promoter region is incorporated into a heterologous expression plasmid and then altered by conventional site-directed mutagenesis (see, e.g., Morinaga, et al., Biotechnology, 2:636 ,1984).
• site-directed mutagenesis see, e.g., Morinaga, et al., Biotechnology, 2:636 ,1984).
• the repressor-encoding sequence is altered in an analogous fashion.
• the resultant mutant repressor/promoter pair is analyzed for its ability to promote secretion of the heterologous protein by comparison of expression with the wild-type plasmid not having been subjected to the site-directed mutagenesis.
• the subject matter disclosed herein provides a drugless or antibiotic-free concept wherein the repressor activity (such as the cl repressor), expressed from the plasmid, inhibits transcription of a toxic gene product (such as the sacB gene product) placed under a ⁇ phage promoter located on the cell host chromosome and wherein the host cells' viability in the presence of a substrate, such as sucrose, is ensured by a sufficient expression level from the plasmid of a repressor protein from the plasmid (such as the ⁇ cl repressor protein).
• a substrate such as sucrose
• plasmid covers any DNA transcription unit comprising a polynucleotide according to the invention and the elements necessary for its in vivo expression in a cell or cells of the desired host or target; and, in this regard, it is noted that a supercoiled or non- supercoiled, circular plasmid, as well as a linear form, are intended to be within the scope of the invention.
• a drugless plasmid comprising one or more repressor gene operably linked to one or more promoter.
• the drugless plasmid comprises a polynucleotide encoding a cl repressor protein.
• the drugless plasmid comprises a promoter operably linked to the polynucleotide encoding the cl repressor protein.
• the promoter may be the native cl gene promoter or the weak promoter of Kanamycin gene.
• the drugless plasmid further comprises a heterologous polynucleotide encoding an immunogen or a protein.
• the drugless plasmid comprises a promoter operably linked to the heterologous polynucleotide encoding the immunogen or protein. Any suitable promoter known in the art can be employed in the drugless plasmids according to the present invention, including bacterial, yeast, fungal, insect, mammalian, and plant promoters.
• the promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig, the Super promoter (Ni, M. et al., Plant J. 7, 661-676, 1995).
• CMV-IE promoter can comprise the actual promoter part, which may or may not be associated with the enhancer part.
• EP-A-260 148 EP-A-323 597, U.S. Patents Nos. 5,168,062, 5,385,839, and
• promoters from gram- negative bacteria are also suitable including, but not limited to, promoters isolated from Avibacterium, Brucella, Escherichia coli, Haemophilus (e.g., Haemophilus suis), Salmonella (e.g., Salmonella enteridis, Salmonella typhimurium, Salmonella infantis), Shigella, Pasteurella, and Rimeirella.
• the plasmids may comprise other expression control elements. It is particularly advantageous to incorporate stabilizing sequence, e.g., intron sequence, for example, the first intron of the hCMV-IE (PCT Application No. WO1989/01036), the intron II of the rabbit ⁇ - globin gene (van Ooyen et al., 1979).
• the plasmids may comprise 3' UTR.
• the 3' UTR may be, but not limited to, agrobacterium nopaline synthase (Nos) 3' UTR.
• polyA polyadenylation signal
• BGH bovine growth hormone
• a "host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector.
• an exogenous polynucleotide such as a recombinant plasmid or vector.
• genetically altered cells the term refers both to the originally altered cell and to the progeny thereof.
• Another embodiment of the present invention relates to a gram-negative bacterium host strain comprising a drugless plasmid.
• One aspect of the embodiment relates to a gram- negative bacterium host train comprising the drugless plasmid and a heterologous polynucleotide inserted in one or more non-essential region of the host chromosome, and wherein the heterologous polynucleotide is operably linked to a promoter which can be tightly regulated by a repressor.
• the heterologous polynucleotide inserted in the host chromosome is a sacB gene.
• the sacB gene is operably linked to a promoter, such as the right promoter of the ⁇ phage.
• a promoter such as the right promoter of the ⁇ phage.
• the sacB gene and the promoter may be inserted in multiple copies, for example, two or three copies, in the host chromosome.
• the non-essential region of the host strain may be the deA or purN gene on the E.coli chromosome.
• the deA and purN genes are nonessential and deletion of the these genes does not affect the growth rate of the bacteria (Kim, J. et al., Biochemistry. 46, 44:12501-12511).
• one copy of the sacB gene and promoter cassette is inserted in either deA locus ox purN locus by allelic replacement.
• two copies of the sacB gene and promoter cassette are inserted in either deA locus ox purN locus.
• two copies of the sacB gene and promoter cassette are inserted in the host chromosome, wherein one copy of the sacB gene and promoter cassette is inserted in the deA locus and the other copy is inserted in the purN locus.
• the sacB gene and promoter cassette refers to the polynucleotide comprising the sacB gene operably linked to a promoter.
• the sacB gene and promoter cassette comprises a sacB gene and the right promoter of the ⁇ phage.
• the sacB gene and promoter cassette comprises a polynucleotide having the sequence as set forth in SEQ ID NO:75.
• the subject matter disclosed herein further relates to a vaccine or composition comprising the drugless plasmids comprising a heterologous gene encoding an immunogen or a protein, or a vaccine or composition comprising the immunogen or protein expressed using the drugless plasmids.
• the vaccine or composition may further comprise a pharmaceutical acceptable carrier.
• the immunogen is selected from a feline pathogen such as, but not limited to, feline herpesvirus (FHV), feline calicivirus (FCV), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), rabies virus, and the like, and combinations thereof.
• a feline pathogen such as, but not limited to, feline herpesvirus (FHV), feline calicivirus (FCV), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), rabies virus, and the like, and combinations thereof.
• the immunogen is selected from a canine pathogen including, but not limited to, rabies virus, canine herpesvirus (CHV), canine parvovirus (CPV), canine coronavirus, Leptospira canicola, Leptospira icterohaemorragiae, Leptospira grippotyphosa, Borrelia burgdorferi, Bordetella bronchiseptica and the like, and combinations thereof.
• rabies virus canine herpesvirus (CHV), canine parvovirus (CPV)
• canine coronavirus canine coronavirus
• Leptospira canicola canine herpesvirus
• Leptospira icterohaemorragiae Leptospira grippotyphosa
• Borrelia burgdorferi Bordetella bronchiseptica and the like, and combinations thereof.
• the immunogen is selected from an equine pathogen, such as equine herpesvirus (type 1 or type 4), equine influenza virus, tetanus, west nile virus, Streptococcus equi, Rhodococcus equi and the like or combinations thereof.
• equine pathogen such as equine herpesvirus (type 1 or type 4), equine influenza virus, tetanus, west nile virus, Streptococcus equi, Rhodococcus equi and the like or combinations thereof.
• the immunogen is selected from a bovine pathogen, such as rabies virus, bovine rotavirus, bovine parainfluenza virus type 3 (bPIV-3), bovine coronavirus, bovine viral diarrhea virus (BVDV), foot and mouth disease virus (FMDV), bovine respiratory syncytial virus (BRSV), Infectious Bovine Rhinotracheitis virus (IBR), Escherichia coli, Pasteurella multocida, Pasteurella haemolytica, salmonella, Cryptosporidium and the like and combinations thereof.
• a bovine pathogen such as rabies virus, bovine rotavirus, bovine parainfluenza virus type 3 (bPIV-3), bovine coronavirus, bovine viral diarrhea virus (BVDV), foot and mouth disease virus (FMDV), bovine respiratory syncytial virus (BRSV), Infectious Bovine Rhinotracheitis virus (IBR), Escherichia coli, Pasteurella multocida, Pasteurella haemolytica
• the immunogen is selected from a porcine pathogen such as, but not limited to, swine influenza virus (SIV), porcine circovirus type 2 (PCV-2), porcine reproductive respiratory syndrome virus (PRRS), pseudorabies virus (PRV), porcine parvovirus (PPV), FMDV, Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Escherichia coli, Bluetongue virus, African Horse Sickness virus, Rift Valley Fever, Nipah virus and the like, and combinations thereof.
• a porcine pathogen such as, but not limited to, swine influenza virus (SIV), porcine circovirus type 2 (PCV-2), porcine reproductive respiratory syndrome virus (PRRS), pseudorabies virus (PRV), porcine parvovirus (PPV), FMDV, Mycoplasma hyopneumoniae, Erysipel
• antigen or "immunogen” is used interchangeably and means a substance that induces a specific immune response in a host animal.
• the antigen may comprise a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a protein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof.
• the immunogen or antigen may comprise a toxin or antitoxin.
• Suitable pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
• the cationic lipids containing a quaternary ammonium salt which are advantageously but not exclusively suitable for plasmids, are advantageously those having the following formula:
• Rl is a saturated or unsaturated straight-chain aliphatic radical having 12 to
• R2 is another aliphatic radical containing 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g. the DMRIE.
• X is an amine or hydroxyl group, e.g. the DMRIE.
• the cationic lipid can be associated with a neutral lipid, e.g. the DOPE.
• DMRIE N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l -propane ammonium; WO96/34109
• DOPE dioleoyl-phosphatidyl-ethanol amine
• the plasmid mixture with the adjuvant is formed extemporaneously and advantageously contemporaneously with administration of the preparation or shortly before administration of the preparation; for instance, shortly before or prior to administration, the plasmid-adjuvant mixture is formed, advantageously so as to give enough time prior to administration for the mixture to form a complex, e.g. between about 10 and about 60 minutes prior to administration, such as approximately 30 minutes prior to administration.
• the DMRIE:DOPE molar ratio is advantageously about 95: about 5 to about 5:about 95, more advantageously about 1: about 1, e.g., 1:1.
• the DMRIE or DMRIE-DOPE adjuvant: plasmid weight ratio can be between about 50: about 1 and about 1: about 10, such as about 10: about 1 and about 1: about 5, and advantageously about 1: about 1 and about 1: about 2, e.g., 1:1 and 1:2.
• pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle may be a water-in-oil emulsion.
• suitable water-in-oil emulsions include oil-based water-in-oil vaccinal emulsions which are stable and fluid at 4 0 C containing: from 6 to 50 v/v % of an antigen-containing aqueous phase, preferably from 12 to 25 v/v %, from 50 to 94 v/v % of an oil phase containing in total or in part a non- metabolizable oil (e.g., mineral oil such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil, or fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % of surfactants, preferably from 3 to 8 p/v %, the latter being in total or in part, or in a mixture either polyglycerol esters, said polyglycerol esters being a mixture either
• surfactants examples include ethoxylated sorbitan esters (e.g., polyoxyethylene (20) sorbitan monooleate (Tween 80®), available from AppliChem, Inc., Cheshire, CT) and sorbitan esters (e.g., sorbitan monooleate (Span 80®), available from Sigma Aldrich, St. Louis, MO).
• ethoxylated sorbitan esters e.g., polyoxyethylene (20) sorbitan monooleate (Tween 80®), available from AppliChem, Inc., Cheshire, CT
• sorbitan esters e.g., sorbitan monooleate (Span 80®), available from Sigma Aldrich, St. Louis, MO.
• sorbitan esters e.g., sorbitan monooleate (Span 80®)
• the antigen-containing aqueous phase comprises a saline solution comprising one or more buffering agents.
• a suitable buffering solution is phosphate buffered saline.
• the water-in-oil emulsion may be a water/oil/water (W /OfW) triple emulsion (see, e.g., U.S. Patent No. 6,358,500, incorporated herein by reference). Examples of other suitable emulsions are described in U.S. Patent No. 7,371,395, incorporated herein by reference.
• the vaccine or composition can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration.
• the route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal).
• the vaccine or composition can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies.
• Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
• the vaccine or composition may be administered as a spray or mixed in food and/or water or delivered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like.
• compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
• Standard pharmaceutical texts such as "Remington's Pharmaceutical Sciences," 1990 may be consulted to prepare suitable preparations, without undue experimentation.
• the subject matter disclosed herein further relates to a method of producing a drugless plasmid comprising the steps of 1) engineering a gram negative bacterium host strain comprising a heterologous polynucleotide inserted by allelic replacement in one or more non-essential region of the host chromosome; 2) constructing a DNA plasmid comprising a polynucleotide encoding a cl repressor protein; 3) transforming the bacterium host strain with the DNA plasmid comprising the gene encoding the cl repressor protein; 4) growing the transformed bacterium host stain in the presence of sucrose at a temperature ranging from 30 0 C to 42°C; and 5) recovering the DNA plasmid.
• the DNA plasmid further comprises a heterologous polynucleotide encoding an immunogen or a protein, wherein the heterologous polynucleotide is operably linked to a promoter.
• the promoter may be a promoter functional in prokaryotic or eukaryotic cell, such as a CMV promoter.
• the subject matter disclosed herein further relates to a method of producing a protein or an immunogen using the drugless plasmids comprising the steps of 1) engineering a gram negative bacterium host strain comprising a heterologous polynucleotide inserted by allelic replacement in one or more non-essential region of the host chromosome; 2) constructing a DNA plasmid comprising a polynucleotide encoding a cl repressor protein and a gene encoding an immunogen or a protein; 3) transforming the bacterium host strain with the DNA plasmid comprising the polynucleotide encoding the cl repressor protein and the gene encoding the immunogen or protein; 4) growing the transformed bacterium host stain in the presence of sucrose at a temperature ranging from 30 0 C to 42°C; and 5) recovering the immunogen or protein.
• the gene encoding an immunogen or a protein is operably linked to a promoter functional in prokaryotic cells.
• the promoter may be promoter from gram-negative bacteria, including, but not limited to, promoters isolated from Avibacterium, Brucella, Escherichia coli, Haemophilus (e.g., Haemophilus suis), Salmonella (e.g., Salmonella enteridis, Salmonella typhimurium, Salmonella infantis), Shigella, Pasteurella, and Rimeirella.
• the heterologous polynucleotide inserted in the host chromosome encodes a sacB protein.
• the sucrose concentration may range from 0% to about 20%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%.
• the sucrose contraction may range from about 1% to about 10%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, or about 9% to about 10%.
• the sucrose contraction may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
• the temperature may range from about 30 0 C to about 42°C. In another aspect, the temperature may range from about 30 0 C to about 41 0 C, about 30 0 C to about 39°C, about 30 0 C to about 38°C, about 30 0 C to about 37°C. In yet another aspect, the temperature may be about 30 0 C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40 0 C, or about 41°C.
• the term recovering includes but is not limited to collecting, extracting, harvesting, or purifying from the culture medium.
• An "isolated" biological component refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organells.
• Polynucleotides (including DNA plasmids) and proteins that have been "isolated” include polynucleotides and proteins purified by standard purification methods, for example, see Russell, David W.; Sambrook, Joseph (2001), Molecular cloning: a laboratory manual. Cold Spring Harbor, N. Y: Cold Spring Harbor Laboratory.
• the term also embraces polynucleotides and proteins prepared by recombinant technology as well as chemical synthesis.
• purified does not require absolute purity; rather, it is intended as a relative term.
• a purified polypeptide or polynucleotide preparation is one in which the polypeptide or polynucleotide is more enriched than the polypeptide or polynucleotide is in its natural environment. That is the polypeptide or polynucleotide is separated from cellular components.
• substantially purified is intended that such that at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed.
• the polypeptide or polynucleotide may be partially purified. By “partially purified” is intended that less than 60% of the cellular components or material is removed.
• the present invention further provides for methods for inducing an immune or protective response in an animal, comprising administering to the animal an immunological composition, a vaccine or a composition according to the invention.
• the immune responses elicited are notably antibody and/or cellular immune responses, and in particular, a gamma- interferon response.
• the present invention provides for methods to immunize against, or to prevent or to reduce the symptoms caused by, infection of an animal with a pathogenic organism (for example, infection by a virus, bacteria, fungus, or protozoan parasite).
• the method of the present invention is useful in vertebrate animals including, but not limited to, humans, canines (e.g., dogs), felines (e.g., cats); equines (e.g., horses), bovines (e.g., cattle) and porcine animals (e.g., pigs), as well as in avians including, but not limited to, chickens, turkeys, ducks, geese, a quail, a pheasant, parrots, finches, hawks, crows and ratites (ostrich, emu, cassowary, and the like).
• canines e.g., dogs
• felines e.g., cats
• equines e.g., horses
• the method of the present invention is also useful for providing fish DNA vaccines.
• these methods consist of the vaccination of pregnant females before parturition by administering a vaccine composition made according to the invention.
• These methods further include the induction of protective antibodies elicited by the vaccination protocol and the transfer of these protective antibodies from vaccinated pregnant females to their offspring. The transfer of such maternal antibodies subsequently protects the offspring from disease.
• the dosage of the vaccine composition made according to the present invention will depend on the species, breed, age, size, vaccination history, and health status of the animal to be vaccinated. Other factors like antigen concentration, additional vaccine components, and route of administration (i.e., subcutaneous, intradermal, oral, intramuscular or intravenous administration) will also impact the effective dosage.
• the dosage of vaccine to administer is easily determinable based on the antigen concentration of the vaccine, the route of administration, and the age and condition of the animal to be vaccinated. Each batch of antigen may be individually calibrated.
• the volume of a dose may be from about 0.1 ml to about 0.5 ml and, advantageously, from about 0.3 ml to about 0.5 ml.
• the volume of a dose may be from about 0.2 ml to about 3.0 ml, advantageously from about 0.3 ml to about 2.0 ml, and more advantageously, from about 0.5 ml to about 1.0 ml.
• the volume of dose may be from about 0.2 ml to about 5.0 ml, advantageously from about 0.3 ml to about 3.0 ml, and more advantageously from 0.5 ml to about 2.0 ml.
• the vaccine composition is administered as a parenteral injection (i.e., subcutaneously, intradermally, or intramuscularly).
• the composition may be administered as one dose or, in alternate embodiments, administered in repeated doses of from about two to about five doses given at intervals of about two to about six weeks, preferably from about two to about five weeks.
• the number of doses and the time interval between vaccinations depends on a number of factors including, but not limited to, the age of the animal vaccinated; the condition of the animal; the route of immunization; amount of antigen available per dose; and the like.
• the period will generally be longer than a week and preferably will be between about two to about five weeks.
• a booster vaccination before or during pregnancy, at about an annual interval may be performed.
• the present invention also contemplates administering a vaccine composition using a needlefree injector such as Pigjet®, Avijet®, Dermojet® or Biojector® (Bioject, Oregon, USA).
• the present invention further relates to a kit comprising a first vial containing an active ingredient such as an immunogen or pharmaceutical composition and, in a second vial, a diluent made according to the present invention.
• the immunogen may be in a lyophilized form, a dried form or in aqueous solution as described herein.
• the following example demonstrates a new concept for maintaining a high number of plasmid copies within a gram-negative host cell which is based on three components: 1. a gram-negative bacterium host which expresses a toxic product for the gram- negative bacterium under defined culture conditions wherein the toxic gene is inserted into one or more non essential region of bacterium host chromosome.
• the gram negative bacterium is transformed to express the sacB gene coding for levansucrase (see FIG. 1).
• the second and third components relate to the cl repressor gene product and the Right ⁇ promoter.
• the cl gene of ⁇ bacteriophage codes for the ⁇ repressor.
• the region of the genome that codes for the cl repressor protein is known as the immunity region.
• the immunity region is illustrated in FIG. 2.
• a plasmid system expressing the cl gene product should inhibit the transcription of the sacB gene placed under the control of the ⁇ promoter. Such a repression should confer resistance to sucrose and allow both the plasmid maintenance and propagation in the host cells (see FIG 1).
• FIG. 3 describes the overview of the "drugless” concept wherein the cl repressor activity, expressed from the plasmid, inhibits transcription of the toxic sacB gene product placed under the ⁇ Pr promoter located on the cell host chromosome and wherein the host E. coli cells' viability in the presence of sucrose is ensured by a sufficient expression level of the ⁇ cl repressor protein from the plasmid.
• the cl repressor binding to its specific promoter is optimal at temperatures between 3O 0 C and 37 0 C (see FIG. 3).
• the cl repressor will not bind the promoter at temperatures higher than 4O 0 C. Therefore, a switch to 42 0 C inhibits the binding activity of cl repressor to the ⁇ Pr promoter and should render the host cells sensitive to sucrose (more particularly the levan subproducts) as a consequence.
• the 42 0 C temperature incubation in the presence of sucrose is a condition ad hoc to validate the functionality of the entire system and constitutes a "negative proof in support of the full functionality of the system (see FIG 4 and FIG 6C).
• the parental cells ⁇ Prr.sacB purN ⁇ .kan transformed with either the pPB829 or pPB838 plasmid growing in the presence of either Cat or Sucrose were not viable when plated on LB sucrose agar upon incubation at
• Two engineered host strains containing the ⁇ Prr.sacB ⁇ kan cassette were prepared wherein the cassette, containing the sacB gene under the control of the lambda promoter ( ⁇ Pr), is marked with Kanamycin and then introduced into the E. coli chromosome by allelic replacement of either edA or purN gene. These genes are not essential and their deletion does not affect the growth rate. The transformed cells became highly sensitive to sucrose.
• FIGS 8A-B show that the ⁇ Prr.sacB ⁇ .kan cassette to be inserted by allelic replacement of edA or purN gene, respectively, into the E. coli chromosome is composed of six independent components that were PCR amplified before being fused by joining PCR.
• FIG 8D shows that the ⁇ Prr.sacB Qkan cassette was inserted into the chromosome of
• E. coli by allelic replacement of the edA ox purN gene.
• This cassette insertion was performed by transforming E. coli with a linear DNA template, containing a deleted gene flanked by up- and downstream homologous region of the chromosome locus, and transferred into a recombination-proficient E. coli strain, such as recD, recB recC she A mutants.
• the E. coli strain for the engineering of the parental strain was the BJ5183 strain from Stratagene (ref# 200154), wherein the genotype was end Al sbcBC recBC galK met thi-l bidl hsdR (Str r ). As shown, the initial phenotype of the E.
• coli wild type was Kan s , Cat s and Sucrose R .
• the phenotype turned Kan R , Cat s and Sucrose s . This latest phenotype was one of the parental strain used for the further experiments dealing with the demonstration of the functionality of the antibiotic-free plasmid.
• FIG 8C shows that the ⁇ Prr.sacB ⁇ .cat cassette to be inserted by allelic replacement of edA gene into the Pr ⁇ ::sacB ⁇ kan E. coli chromosome was composed of six independent components that were PCR amplified before being fused by joining PCR.
• the goal of the new ⁇ Prr.sacB ⁇ .cat cassette targeting the deletion of the edA gene by allelic exchange was to engineer a double deletion mutant AedA ApurN expressing two sacB cassettes.
• FIG 8E shows that when the parental strain was transformed with the cl plasmid (pPB829 or pPB838), the cells (parental strain + cl plasmid [pPB829 or pPB838]) became resistant to kanamycin (Kan R ), chloramphenicol (Cat R ) and sucrose (Sucrose R ).
• the figure illustrates the coexistence of the engineered host strain (ApurN ⁇ Pr::sacB + ⁇ kan) or (AedA ⁇ Pr: : sacB + ⁇ kan) and drug plasmid to demonstrate proof of concept when the sacB gene was placed under the control of the lambda promoter and the construct was introduced into the chromosome of E.
• the ⁇ Promoter ( ⁇ Pr) was amplified with PB 1232 and PB 1233 primers for purN deletion.
• PB1232 primer SEQ ID NO:6
• PB1234 (SEQ ID NO:8):
• ⁇ Promoter SEQ ID NO:5
• pLDR8 plasmid ATCC# 77357
• the sacB gene was amplified with the PB 1192 (SEQ ID NO:9) (ATGGATATGAACATCAAAAAGTTTGC) and PB1193 (SEQ ID NO:10)
• the Ua promoter was amplified with the PBl 194 (SEQ ID NO:11) (ATGTGCGCGGAACCCCTATTTG) and PB1195 (SEQ ID NO:12) (GACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATTGAAGC) primers using the pCMV ⁇ as DNA template.
• the Kanamycin resistance gene was amplified with the PB1196 (SEQ ID NO:13) (ATGAGCCATATTCAACGGGAAACG) and PB1197 (SEQ ID NO:13) (ATGAGCCATATTCAACGGGAAACG) and PB1197 (SEQ ID NO:13) (ATGAGCCATATTCAACGGGAAACG) and PB1197 (SEQ ID NO:13) (ATGAGCCATATTCAACGGGAAACG) and PB1197 (SEQ ID NO:13) (ATGAGCCATATTCAACGGGAAACG) and PB1197 (SEQ ID NO:13) (ATGAGCCATATTCA
• the 5' flanking region of the purN gene was amplified with PB1237 (SEQ ID NO:15) (TTTGCGGCCGCTGGTGGTGGTCGCCATGTGCGTTAATGACC) and PB 1199 (SEQ ID NO:16) (TATTCGATAACCACGCGTTGTTCGG) using genomic DNA of the E. coli SCSI strain as DNA template.
• the 3' flanking region of the purN gene was amplified with PB1200 (SEQ ID NO: 17) (GCGCCAGCTCTGCCGCTGGCGTTTTTC) and PB 1238 (SEQ ID NO: 18) (TTTGGATCCGCTGGTGGATATCATCAAGGCAGTAACGCAGAATG) primers using genomic DNA of the E. coli SCSI strain as DNA template.
• the 5' flanking region of the edA gene was amplified with the PB1235 (SEQ ID NO:19) (TTTGCGGCCGCTGGTGGTTGAGAACCAGGTGATTGAAGCGCC) and PB 1209 (SEQ ID NO:20) (CTCTCGCCTGATTACAAATTTGTCGTC) primers using genomic DNA of SCSI as DNA template.
• the 3' flanking region of the edA gene was amplified with the following primers PB1210 (SEQ ID NO:21) (AGGATCGGGCATTTTTGTAGCGT) and PB1236 (SEQ ID NO:22) (TTTCTAGAGCTGGTGGCGACTACCGTGAATCCTGGCAACC) primers using plasmid pLDR8 as template DNA.
• PCR condition was as follows: PCR mixture (final 25 ⁇ l), 1 ⁇ l of each PCR fragment, 2 ⁇ l of dNTP (2.5 mM each, 5 ⁇ l of 5xPCR buffer, 11.5 ⁇ l sterile distilled water, 0.5 ⁇ l Phusion DNA polymerase (Finenzymes, Finland); PCR cycles: 98 0 C 30s, (98 0 C 10 s, 6O 0 C 30 s, 72 0 C 5 min)* 15 cycles and 72 0 C 10 min.
• the third round PCR amplification was performed with 1 ⁇ l of the purified second round product by using the PB 1235 and PB 1236 primers fox purN (allelic replacement) and the PB 1237 and PB 1238 primers for edA (allelic replacement).
• PCR condition was as follows: PCR mixture (final 50 ⁇ l), 1 ⁇ l of second PCR fragment, 1 ⁇ l of dNTP (2.5 mM each, 10 ⁇ l of 5xPCR buffer, 37 ⁇ l sterile distilled water, 0.25 ⁇ l forward primer, 0.25 ⁇ l reverse primer, 0.5 ⁇ l Phusion DNA polymerase (Finenzymes, Finland); PCR cycles: 98 0 C 30s, (98 0 C 15 s, 6O 0 C 30 s, 72 0 C 4 min)* 35 cycles and 72 0 C 10 min.
• the final PCR-joined amplicons (4007 bp for the purN allelic replacement and 3407 bp for the edA allelic replacement) were checked on agarose gel and purified before transformation into E.coli. Each joined-PCR amplicon was checked by restriction analysis.
• the linear DNA fragment ⁇ Prr.sacB ⁇ .kan) encoding a sacE gene placed under the control of ⁇ Pr promoter (SEQ ID NO:75) and Kan resistance gene placed under the control of the bla promoter flanked by two long regions homologous to the DNA sequences bordering the target locus ⁇ edA or purN) was integrated into the chromosome of E. coli by electroporation.
• Approximately 300 transformant candidates (ApurN ⁇ Prr.sacB ⁇ .kan or ⁇ edA ⁇ Prr.sacB ⁇ .kan single mutant) were obtained on LB agar plate containing kanamycin as selection pressure.
• PB 1204 SEQ ID NO:23
• PB 1205 SEQ ID NO:24
• CCAGCCACGCGGCGTTTTCGTGC CCAGCCACGCGCGGCGTTTTCGTGC
• GGTGGATGGCGTCCATTTCTGTGC GGTGGATGGCGTCCATTTCTGTGC
• PB 1196 primers was performed to verify the right and correct insertion of the cassette at the edA locus.
• the PCR using the PB1212 (SEQ ID NO:28) (CAAAAGTGTTAAGCGGTAACCTG) and PB1233 primers was performed to verify the left and correct insertion of the cassette at the edA locus.
• the PCR using PB 1203 and PBl 196 primers was performed to verify the right and correct insertion of the cassette at the purN locus.
• the PCR using the PB 1202 and PB 1233 primers was performed to verify the left and correct insertion of the cassette at the purN locus (see FIG 9C).
• the linear DNA fragment ( ⁇ Prr.sacB ⁇ .cat) encoding a sacB gene (placed under the control of ⁇ Pr promoter) and Cat resistance gene (placed under the control of its natural cat promoter) flanked by two long regions homologous to the DNA sequences bordering the target locus (edA) was integrated into the chromosome of the ApurN ⁇ Prr.sacB ⁇ .kan E. coli strain by electroporation. Approximately 200 transformant candidates (ApurN ⁇ Prr.sacB ⁇ .kan ⁇ edA ⁇ Prr.sacB ⁇ .cat double mutant) were obtained on LB agar plate containing chloramphenicol as selection pressure.
• FIG. 8E illustrates host strain (one sacB cassette [ ⁇ Pr::sacB ⁇ .kan]) and drugless plasmid coexistence.
• the initial E. coli genotype is Kan s , Cat s , Sucrose R .
• Cassette integration into the chromosome of E. coli ApurN Pr ⁇ rsacB ⁇ Kan generated a new genotype, Kan R , Cat s , Sucrose s .
• Introduction of the plasmid harboring the cl repressor generated the final genotype, Kan R , Cat R , Sucrose R .
• Example 3 Generation of Plasmids Harboring the cl Repressor Expression
• pPB828-derived plasmids such as pPB829 and pPB844-pPB847 were generated containing the cl ORF of ⁇ phage, under the control of either its own promoter or the weak promoter of the kanamycin gene (Pl).
• the pPB838 plamid containing cl gene placed under the control of the weak promoter of Kanamycin gene (Pl) was generated using an intermediate plasmid commercial vector pMCS5 in which chloramphenicol acetyl transferase gene (cat) replaced the ampicillin resistance gene. (FIG 19).
• the pPB844 and pPB845 plasmids contain the cl gene (placed under the control of the Pl promoter) cloned into the pPB828 vector which is a pVR1012 derivative (pVR1020 or 1012 plasmid, VICAL Inc.; Luke et al., 1997; Hartikka et al., 1996, see, e.g., U.S. Patent Nos. 5,846,946 and 6,451,769) containing cat gene.
• the plasmids differ in the orientation of the cl gene into the plasmid (FIG 20-21).
• the pPB846 and pPB847 plasmids contain the cl gene (placed under the control of its own (native) promoter SEQ ID NO: 30) cloned into pPB828 vector which is a pVR1012 derivative containing cat gene.
• the plasmids differ in the orientation of the cl gene in the plasmid (FIG 22-23).
• the pPB885 was prepared from the pPB838 plasmid [cl gene placed under the control of the weak promoter of Kanamycin gene (Pl)] in which the chloramphenicol acetyl transferase gene (cat) was removed by Avrll restriction digestion (FIG 24).
• the pPB829-derived plasmids such as pPB896 contains cl gene placed under the control of the weak promoter of Kanamycin gene (Pl).
• the pPB896 plasmid contains the GFP marker gene placed under the control of the eukaryotic CMV promoter (FIG 25).
• plasmids were used as vectors for cloning a heterologous polynucleotide, a gene of interest (transgene) in the drugless system.
• Each plasmid harbors the antibiotic resistance gene (cat) that can be excised and removed by a unique restriction enzyme to allow the plasmid propagation in the appropriate E. coli sacB + strain in the presence of sucrose without antibiotic selection pressure (such as chloramphenicol).
• the antibiotic resistance gene (cat) on these plasmids was used only for proof of concept.
• the cat gene is finally removed in the final drugless plasmids (see FIGs 13-15 and Example 4).
• the cl gene sequence from plasmid pLDR8 produces a repressor protein cI857 mutant (NCBI: AB248924, Cloning vector pND707,Love, CA. et al., Gene. 17; 176(l-2):49-53; 1996)
• pPB828 plasmid (pVR1012-based plasmid + Cat gene) (FIG 17)
• the pPB828-derived plasmids such as pPB829 was generated containing the cl ORF of ⁇ phage, under the weak promoter of the kanamycin gene (Pl) and the Cat gene that is excisable by a unique EcoRI digestion site.
• a pVR1012-based expression vector was generated containing the chloramphenicol acetyl transferase (cat) gene giving rise to the pPB828 plasmid.
• the cat gene from pPB627 was a derivative from pNB335 (Merial proprietary material) which is itself a derivative from initial plasmid pSW23T (cloning plasmid for GenBank Accession No. AY733066).
• the DNA fragment corresponding to the cat gene (798 bp) (SEQ ID NO:31 for DNA, SEQ ID NO:32 for protein) was obtained by PCR using the primers PB 1184 (SEQ ID NO:33) (GAATTCCGGTCCGGGCGAAAATGAGACGTTGATCGGCS) [Containing an Ecorl site (underlined)] and PB 1185 (SEQ ID NO:34)
• the DNA fragment corresponding to the Cat gene was obtained by enzyme digestion of pCRblunt/PBl 184-1185 plasmid with EclB ⁇ ll and EcoRV.
• the 897 bp fragment was ligated into the pVR1012-based plasmid, previously digested with Mscl and Stul (3303 pb) to generate plasmid pPB828 (FIG 17).
• the identity of pPB828 was confirmed by restriction analysis ⁇ PvuII and Ncol digestion to determine the size of the insert and EcoRI to verify that the gene cat is well removed).
• pPB829 plasmid (pPB828 plasmid + Plr.clgene) (FIG 18)
• the construct containing the cl gene under Pl promoter was obtained with a fusion PCR.
• the DNA fragment corresponding to the Pl weak promoter was obtained by PCR using primers PB 1186 (SEQ ID NO:35) (TCATACCAGGCCTAGGTGATACGCCTATTTTTATAGGTTAATG) and PBl 187 (SEQ ID NO:36) (AACACCCCTTGTATTACTGTTTATG), using pLL14 (see Merial patent application US2005/0164946) as a template and Phusion DNA polymerase.
• the DNA fragment corresponding to the cl gene was obtained by PCR using primers PBl 188 (SEQ ID NO:37) (AATACAAGGGGTGTTATGAGCACAAAAAAGAAACCATTAACAC) [containing a complementary region of Plsequence at 5' end (underlined)] and PBl 189 (SEQ ID NO:38) (CCGGAATTCGGCGCGTCAGCCAAACGTCTCTTCAGGCCACTG) using pLDR8 (ATCC # 77357) as a template and Phusion DNA polymerase.
• the two PCR products (respectively 151 and 729 bp) were purified and used as template in a second PCR step with the PBl 186 and PBl 189 primers and the Phusion DNA polymerase (Finnzymes, Finland)
• the two PCR products (respectively 151 and 729 bp) were purified and ligated with plasmid pPB828 digested with Ascl and Avrll to generate plasmid pPB829 (4970 bp) (FIG 9A).
• the ligation was set up with the "In-Fusion PCR Cloning Kit” (cat. No. 740590.250) from Clontech (Takara Bio Europe, St-Germain-en-Laye,France).
• Clones pPB829 were selected following a HindII digestion analysis The integrity of the sequence of cl gene and Pl promoter was confirmed by sequencing the plasmid pPB829.
• pPB896 plasmid (pPB829 plasmid + Plr.clgene + GFP) (FIG 25)
• the GFP marker gene was placed under the control of the eukaryotic CMV promoter (SEQ ID NO:43).
• the GFP gene was prepared from restriction digestion of the pCG105 plasmid (pPB828-derived plasmid having the GFP gene placed under the control of the eukaryotic CMV promoter, Merial proprietary material) with both Notl/Bglll. This digested GFP gene was cloned into the pPB829 plasmid previously cut by Notll BgIII giving rise to the pPB896 plasmid.
• the DNA fragment corresponding to the Cat gene was obtained by PCR using the primers PB 1182 (SEQ ID NO:39)
• PCR primers were designed based on the cat sequence of pNB350. Three independent
• PCR products (779 bp) were pooled and ligated into the pCRII vector to obtain plasmid pCRII + PBl 182-1183 (4734 bp).
• Clones of pCRII + PBl 182-1183 were selected following a
• Fragments A and B were ligated to generate plasmids pPB837.1 (3747 bp).
• Plasmid pPB837.2 was generated which does not contain bla promoter region.
• Plasmid pPB837.1 was digested with Nael and Xmnl and fragment Nael-Xmnl of 3300 bp was isolated and purified from agarose gel (fragment A). The fragment A was ligated to obtain the pPB837.2 plasmid (3300bp). Clone pPB837.2 was selected following a BgII digestion.
• Kanamycin resistance gene was obtained with a fusion PCR.
• the DNA fragment corresponding to the Pl weak promoter was obtained by PCR using primers PB 1186 and PBl 187, the pLL14 as a template and Phusion DNA polymerase.
• the DNA fragment corresponding to the cl gene was obtained by PCR using primers PBl 188 and PB 1189, the pLDR8 (ATCC # 77357) as a template and Phusion DNA polymerase (Finnzymes, Finland).
• the two PCR products (respectively 151 and 729 bp) were purified and used as templates in a second PCR step with the PB 1186 and PB 1189 primers and the Phusion DNA polymerase.
• pPB885 plasmid (pMCS5 + Cat without bla promoter) + Pl::cl (FIG 24) This plasmid was deprived from pPB838 plasmid where the chloramphenicol acetyl transferase gene (cat) was removed by Avrll restriction digestion (FIG 24).
• pPB885 plasmid contains the cl gene under the control of the weak promoter of kanamycin gene (Pl).
• the pPB885 plasmid was isolated as described in the example 4 (see below) following transformation of the ApurN ⁇ Pr::sacB Qkan E. coli host strain by direct selection on LB agar plate supplemented with 10% sucrose. plasmids pPB844-pPB845 (FIGS 23-24)
• the DNA fragment corresponding to the cl gene under Pl promoter was obtained by PCR using primers PBl 186 and PB1263 (SEQ ID NO:41)
• the pCRblunt + PBl 186-1263 plasmid was digested with EcoRI and the extremities were filled with Klenow Polymerase to generate a blunt fragment (929 bp : fragment A).
• the pPB828 plasmid was digested with Xmnl and the resulting Xmnl-Xmnl fragment was purified (fragment B: 4135 bp). Fragments A and B were ligated to generate plasmids pPB844 or pPB845. Candidates were screened by a BamHI/Hindlll digestion. In the pPB844 plasmid (see FIG.
• the cl gene is in the same direction as the transgene of interest to be cloned into the MCS (multiple cloning site) of the vector under the control of the CMV promoter (SEQ ID NO:43).
• the cl is oriented in the opposite direction.
• PB1266-PB1263 777 bp
• PB1266-1263 4296 bp
• Clones of pCRblunt + PB1266-1263 were selected following a HindIII digestion.
• the pCRblunt + PB 1266- 1263 plasmid was digested with EcoRI and the extremities were filled with Klenow Polymerase to generate a blunt fragment (842 bp : fragment A).
• pPB828 plasmid was digested with Xmnl and fragment Xmnl-Xmnl was purified (fragment B: 4135 bp). Fragments A and B were ligated to generate plasmids pPB846 (4999 bp) or pPB847. Candidates were screened by a BamHII HindIII digestion. In the pPB846 plasmid (see FIG.
• the cl gene is in the same direction as the gene of interest to be cloned into the MCS site of the vector under the control of the CMV promoter.
• the cl gene is oriented in the opposite direction.
• FIG. 7 shows the constructs of pPB838 and pPB829 harboring the cl repressor that were used to prepare the drugless plasmid. Both plasmids were marked with the Chloramphenicol (Cat R ) gene. The Cat gene on these plasmids were required only for determining proof of concept but can be finally removed (see FIGs 13-15 and Example 4).
• Example 4 Transformation of the E. coli host strain with the cl plasmid(s) and direct selection on a sucrose agar plate
• An experimental procedure that enables the screening of the transformed cells by the chloramphenicol (Cat) marked cl plasmid(s) without the use of antibiotic as selection pressure on agar plate(s) has been efficiently demonstrated (see FIG 13-15).
• Competent cells of E. coli ⁇ Pr::sacB + ⁇ purN ⁇ .Kan were transformed with 1 ⁇ g of either pPB838 or pPB829 plasmid and incubated for 3 hours at 3O 0 C in LB liquid broth medium. 100 ⁇ l aliquot of diluted (10 ⁇ 3 and
• 10 "4 ) transformed cells (with pPB838 and pPB829 plasmids) were spread onto LB agar plate containing 10% sucrose. These dilutions ranging from 10 "3 to 10 "4 appear appropriate to select true transformant cells (e.g. having cl plasmid) since 98-100 % of sucrose resistant cells growing on LB agar plate containing sucrose were Cat R . This suggests that these transformed cells have the cl plasmid (pPB829 or pPB838).
• FIG 13A shows that competent cells of E. coli ⁇ Pr::sacB + ⁇ purN ⁇ .Kan were transformed with 1 ⁇ g of either pPB838 or pPB829 plasmid and incubated for 3 hours at 3O 0 C in LB liquid broth medium before dot-spotting (4 ⁇ l for each dot).
• the figure shows the Log dilutions (ranging from undiluted to 6 Log dilution) on LB agar plates containing either chloramphenicol (15 ⁇ g/ml) or sucrose (10%).
• the untransformed competent parental strain cells of E. coli ⁇ Pr::sacB + were used as standard cells grown under similar condition.
• FIG 13B shows that 100 ⁇ l of transformed (with pPB838 and pPB829 plasmids) and untransformed standard cells were spread on selection LB agar plate containing either cat or sucrose using appropriate Log dilution to determine the rate of transformation efficacy on cat and sucrose and standard spread on sucrose to better define the baseline of spontaneous mutant rate to be considered for successful screening of true transformant cells capable of growth on LB sucrose agar plate.
• spontaneous mutant resistant to sucrose were not detectable when the 10 "3 dilution of transformant cells were spread on LB agar plate containing sucrose.
• FIGS 14A-C illustrate the efficacy of the experimental procedure to select transformed cells with the cl plasmid only (pPB829 and pPB838) without the use of antibiotic as selection pressure.
• FIG 14A shows the experimental overview using replica plate assay to validate the screening of true transformant cells having the cl plasmid. Similar to what is shown in FIG 13B, parental competent cells ⁇ Pr::sacB + ApurN ⁇ .Kan were transformed with the cl plasmid (pPB829 and pPB838) and appropriate Log dilutions were spread onto selective (Cat or Sucrose) LB agar plates before incubation for 3 days at 3O 0 C.
• FIG 14B shows that experimental results confirmed that the Cat R CFUs were identical between the number obtained on sucrose LB agar plates and on Cat/sucrose LB agar plates after replica plate assay.
• the CatR CFUs are i) all sucrose resistant and ii) all resistant to the combination of Cat and Sucrose, signifying that all parental cells following transformation harbor the cl plasmids.
• Panel C shows that the 10 "3 and 10 "4 dilutions of transformant cells selected directly on sucrose were also resistant to sucrose and to the combination of both sucrose and Cat, also signifying that these sucrose resistant CFUs correspond to true transformed cells containing the cl plasmid.
• sucrose resistant CFUs when replicated on a combination of sucrose and Cat did not grow as expected because the chloramphenicol resistance is a phenotypical trait brought by either the pPB829 or pPB838 plasmid.
• FIG 14C represents the statistical data obtained from the replica plate assay (see FIG 14B) and more particularly the percentage of the colonies having the cl plasmid that were selected without the use of antibiotic. Under this experimental condition, the percentage of colonies having the cl plasmid ranges from 98% to 100%.
• Example 5 Different culture conditions with and without sucrose showing that the cl plasmid can be maintained E. coli cells expressing sacB, ⁇ purN ⁇ Pr::sacB + ⁇ .kan were grown in the presence or absence of sucrose in order to check the functionality of the ⁇ Pr::sacB + cassette inserted into the chromosome of E. coli.
• FIG. 4 describes the "sucrose sensitivity" of the inactivated system wherein a switch to 42°C in temperature inhibits the binding activity of cl repressor to the ⁇ Pr promoter and renders the host cells sensitive to sucrose.
• the cl repressor protein expressed from the plasmid, inhibits the transcription of the toxic sacB gene product placed under the ⁇ Pr promoter that is located on the host cell chromosome.
• the host E. coli cells' viability in the presence of sucrose is ensured by a sufficient expression level from the plasmid of the ⁇ cl repressor protein.
• FIGs 6 A, 6B, and 6C The experimental plan for the proof of concept is further illustrated in FIGs 6 A, 6B, and 6C.
• FIGs 10-11 provide results confirming that the genetically engineered host strains were highly resistant to sucrose at 30-37 0 C, while no growth was observed at 42°C.
• FIG. 10 shows that second passage with the E. coli host strains, ⁇ purN ⁇ Pr: :sacB + ⁇ kan, harboring either the pPB829 plasmid or pPB838 plasmid grew well in the presence of sucrose at 32 0 C.
• the sacB gene expression was perfectly repressed by the cl gene product synthesized from both c/plasmids.
• the plasmid maintenance was 100% efficient (versus control on Cat R ) and cells were able to survive in the presence of sucrose.
• the cl gene product was inactivated.
• the plasmid could not be maintained and the cells died in the presence of sucrose. This demonstrates that the cells viability in the presence of sucrose was due to the presence of a functional cl plasmid.
• Example 6 Culture conditions showing that the cl plasmids are stable over 5 passages, without antibiotic selection pressure, and lack of spontaneous mutants for sucrose resistance Once the drugless plasmids were produced, they were tested for stability and their robustness over several passages in fresh broth medium containing either sucrose or Cat.
• FIG. 11 illustrates the stability of the pPB829 and pPB838 plasmids in growing cells, including lack of obvious genetic rearrangement during selection on either chlorampenicol or sucrose. Comparable plasmid yields and maintenance in cultures growing in the presence of sucrose and chloramphenicol were observed even after five successive passages. The antibiotic and non antibiotic systems were at least equivalent in terms of plasmid maintenance and selection from growing cells.
• FIG. 12 illustrates the lack of spontaneous mutations in the ⁇ Prr.sacB cassette inserted in the parental E. coli strain when cells were growing at 3O 0 C. This observation clearly demonstrates the cl plasmid stability in such E. coli genetic background when cells were growing in presence of sucrose (see FIG 10).
• This mutation rate experiment was performed with the parental strain (purN deleted expressing the sacB gene). It was shown that no mutation occurred at 3O 0 C after 18h incubation.
• Clonal cultures of parental cells harboring the sacB + cassette in their chromosome were grown in LB containing sucrose. Cells were harvested at an OD of 1.0 and aliquots of 100 ⁇ L were spread onto LB agar plates containing sucrose.
• Spontaneous mutation occurred at a rate of 1 mutation/1.3 X 10 5 cells after 36 hours when incubated at 37 0 C.
• the cl plasmid maintenance in the cells growing in the presence of sucrose was effective after overnight incubation at 30°C while no growth of the mother strain was detected.
• Example 7 Improvement of the Parental E. coli host strain to reduce the rate of the spontneaous mutation to sucrose by adding a second sacB cassette (AedA ⁇ Pr::sacB ⁇ .cat) into the aforementioned one sacB cassette E. coli host strain (ApurN ⁇ Prr.sacBQka ⁇ ).
• FIG 8C and FIG 9D illustrate how the new parental host strain of E. coli harboring two sacB cassettes (ApurN ⁇ Prr.sacBQkan AedA ⁇ Pr::sacB ⁇ .cat) were engineered.
• FIG lOB-C demonstrate that the dual sacB cassettes E. coli strain (ApurN ⁇ Pr: : sacB + ⁇ kan AedA ⁇ Pr::sacB + ⁇ Cat) was highly sensitive (no growth) in the presence of sucrose when incubated at 3O 0 C and 42 0 C while some spontaneous mutants appears with the one sacB cassette E. coli strain plated on even 10% sucrose (ApurN ⁇ Pr: :sacB ⁇ .kan).
• FIG 1OC also illustrates that the lowest sucrose concentration required(2% final) for the dual sacB cassettes E. coli strain compared to the one sacB cassette E. coli strain (10% final). Moreover, it appears that the two sacB cassettes E.
• FIG 12B-C demonstrate that the presence of two sacB cassettes (rather than one) confers a better robustness to sucrose sensitivity (still optimal at 2% sucrose) at temperatures ranging from 3O 0 C to 37 0 C.
• FIG 12B-C and Tables 3-7 below show that the presence of one sacB cassette in the E. coli chromosome was less robust at sucrose concentration ranging from 2 to 4% at similar temperatures such as 3O 0 C and 37 0 C. As confirmed in this plating assay, the mutation rate to sucrose with the two sacB cassettes E.
• Example 8 Plasmid maintenance and yields obtained in the absence of antibiotic selection pressure (the cl plasmid is maintained at a high copy number in the host strain)
• E. coli host cells expressing sacB and harboring the cl plasmid were grown in the absence of antibiotic selection pressure (e. g. in presence of sucrose) or in the presence of Cat as control.
• the plasmid yield was determined at passages 2 and 5. Plasmids were extracted from cultures maintained at 3O 0 C and subjected to gel electrophoresis. The HindII restriction digestion indicates that no obvious genetic rearrangement occurred in either pPB829 or pPB838 during propagation and selection with either Cat or Sucrose. Moreover, similar cl plasmids maintenance was observed during the selection of the cl plasmids under either Cat or Sucrose selection pressure.
• FIGs. 15A-B illustrate the stability and efficacy of the cl plasmids (pPB829 and pPB838, containing one copy of the sacB cassette) maintenance in cultures, including lack of genetic rearrangement after selection on either chlorampenicol or sucrose. Efficient plasmid maintenance and the stability of cl plasmids grown in the presence of either sucrose or chloramphenicol were observed. Moreover the plasmid yield (copy number) was much higher (approximately 700 per cell) when parental host strain transformed with either the pPB838 or pPB829 were grown at 3O 0 C in the presence of sucrose as selection pressure than cultivated in the presence of Cat as the selection pressure.
• the plasmid maintenance and yield were highly efficient and the copy number was twice higher compared to cells cultivated in the presence of Cat.
• This better cl plasmid yield when host cells were cultivated in the presence of sucrose was due to the weak promoter (Pl) that controls the cl expression.
• the expression of the cl repressor under the control of the weak promoter of Kanamycin gene (Pl) had a positive effect on the plasmid yield to better counteract the toxicity of the sacB gene placed under the control of the ⁇ Pr promoter.
• FIG 15B demonstrates that the plasmid yield (copy number) was much higher
• Example 9 Plasmid maintenance and yields in the absence of antibiotic gene in the cl plasmid: comparative analysis between both one and two sacB cassettes E. coli strains
• Figures 15C-E illustrate the stability and yield of cat-free pPB885 plasmid in both one and two sacB cassettes E. coli strains ⁇ ApurN ⁇ Pr::sacB + ⁇ kan and ApurN ⁇ Pr: :sacB + ⁇ kan AedA ⁇ Pr::sacB + ⁇ cat, respectively).
• Both ApurN ⁇ Pr::sacB km (one sacB cassette) and ApurN ⁇ Pr: :sacB km Aeda ⁇ Pr: :sacB cat (two sacB cassettes) parental strains were transformed with the cl cat-free pPB885 plasmids and incubated for 3 hours in LB prior to plating on sucrose plate and subsequent cultivation (3 passages) in LB liquid growth medium supplemented with sucrose.
• the plasmid yield was determined at passages 3 in LB supplemented with 5% and 10% sucrose for the two and one sacB cassettes E. coli strains, respectively.
• the pPB885 plasmids were extracted from cultures maintained at 37 0 C and subjected to gel electrophoresis.
• the PvuII restriction digestion indicates that no obvious genetic rearrangement occurred in either pPB885 during propagation and selection with Sucrose (see FIG 15D).
• no obvious genetic rearrangement occurred after selection with sucrose at either 10% and 5% in both E. coli host strains, one and two sacB cassette, respectively.
• Figure 15E illustrates the yield of the cat-free pPB885 plasmid in both one and two sacB cassettes E. coli strains (ApurN ⁇ Pr::sacB + ⁇ kan and ApurN ⁇ Pr::sacB + ⁇ kan AedA ⁇ Pr: : sacB + ⁇ cat, respectively).
• the plasmid yield (copy number) was much higher (approximately 278 per cell) in the two sacB cassettes compared to the plasmid yield (approximately 142 per cell) obtained from the one sacB cassette E. coli strain.
• the plasmid yield obtained in this experiment appears lower than the plasmid yield obtained in the previous comparative study (see FIG 15B).
• Example 10 Antibiotic-free cl plasmid transfection in CHO cells
• FIG 15F and Table 8 below illustrates the plasmid transfection efficacy (GFP expression) in CHO cells of the antibiotic-free plasmid (pPB896 /Sucrose as selection pressure) and antibiotic plasmid (pCG105 / Cat as selection pressure). No obvious difference is observed in terms of expressed GFP protein between these two selection pressures (Sucrose versus Cat as antibiotic).
• the pCG105 plasmid (pPB828-derived plasmid having the GFP gene placed under the control of the CMV promoter [Merial property]) and the pPB896 (pPB829 plasmid + Pl::cl gene + GFP) plasmids were used in CHO cells' transfection to evaluate the functionality of the antibiotic-free plasmid concept versus current antibiotic plasmid.
• This experiment was performed using the plasmids isolated from culture of E. coli host strain having one sacB cassette.
• the pCG105 was propagated in E. coli using Kanamycin as selection pressure while the pPB896 was propagated in E. coli using 10% sucrose as selection pressure. DNA plasmids were extracted and dosed at the same concentration.
• FIG 15F illustrates the in vitro expression of the GFP protein encoded by pCG105 and pPB896 after transient transfection of CHO-Kl cells, using Lipofectamine 2000.
• CHO-Kl cells at 90% confluency in 6 cm diameter plates were transfected with 5 ⁇ g plasmid and 10 uL Lipofectamine each, according to manufacturer's instruction. After the transfection, cells were cultivated in MEM-glutamax medium containing 1% SVF for 24 hours. Culture were harvested and analyzed using fluorescence microscopy (FIG 15G) and Flow Cytometry (FACS Calibur [Becton Dickinson]) (Table 8).
• FlG 15F shows that similar GFP epifluorescence was visualized in both transfected CHO-Kl cells.
• the pCG105 and pPB96 plasmids under the selection pressure of chloramphenicol and sucrose were able to transfect the CHO-Kl cells with comparable efficiency.
• Table 8 shows the efficacy of CHO transfection with the pCG105 and pPB896 plasmids. As indicated in this table, a comparable % of CHO cells expressing the GFP protein was determined when CHO was transfected with pCG105 (59% GFP expressed) and pPB896 (48% GFP expressed). These results validated the use of the sucrose-based antibiotic-free plasmid system for application in CHO transfection and DNA vaccine purposes, ultimately.

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